Our basic research on the metabolism of membrane phospholipids will continue in three major areas; 1) purification of the enzymes involved, 2) study of their mechanism of action, and 3) based on the information obtained in the major areas of research, we shall also study their function within the organized membrane system. These studies will center around the phospholipases, but will also involve other enzyme systems of phospholipid metabolism. First we will continue purification of the lysosomal phospholipases A1 and A2, the plasma membrane phospholipase A1, and the mitochondrial phospholipase A2. The techniques to be employed are salt and pH precipitation, ion exchange and affinity chromatography, gel filtration, and isoelectric focusing. Second the mechanism of action of these enzymes will be studied together with the study of the physical-chemistry of the substrates which are water insoluble. We shall use mono-layer and microelectrophoresis techniques to investigate the nature of the lipid changes in the presence of enzyme activators or inhibitors such as metal ions, reaction products, and lipophylic pharmacologic agents. The interaction of these enzymes with the substrates will determine the physical changes that occur during hydrolysis. We plan to utilize our knowledge of the phospholipases by investigating the function of the enzymes within organized systems. We will study the relationship of the phospholipase to mitochondrial respiratory control and membrane charge, as demonstrated by flourescent probes. The role of the lysosomal enzymes in hydrolysis of phagocytized particles and in the fusion of lysosomes and degranulation will be expanded. The function of the plasma membrane phospholipase as it is activated by heparin will be studied in relation to the uptake of lipids via hydrolytic or transacylation reactions. BIBLIOGRAPHIC REFERENCES: Waite, M., Sisson, P., Freude, K.A., and Zieve, F.J.: The hydrolytic and transacylation activity of the phospholipase A1 purified from human post-heparin plasma. Biochem. Biophys. Res. Comm. 67:471-477 (1975). Griffin, H.D., Franson, R., and Waite, M.: Purification and characterization of lysosmal phospholipase A1 from rat liver, Abstr., Fed. Proc. 33:1469 (1974).